大白菜抗根肿病基因定位及连锁标记挖掘
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摘要
为了明确抗根肿病大白菜材料G6所含抗病基因,开发连锁标记,以高感根肿病的大白菜高代自交系G4、高抗根肿病的大白菜高代自交系G6、G4和G6杂交得到的F_1及F_1自交构建的F_2分离群体为材料,通过人工接种、表型鉴定和遗传学分析,发现该抗病材料中的根肿病抗性由显性单基因控制。进一步扩大F_2群体数量对根肿病抗性基因的初定位,筛选与其连锁的分子标记,利用JoinMap 4.0软件对多态性标记进行连锁分析,获得了5个与大白菜抗根肿病基因连锁的InDel标记,其中最近的两侧标记为BrID10727和BrID10867,与抗病基因的遗传距离分别为4.6,2.5 cM,抗病基因定位在大白菜染色体A08上。此外,经验证发现基于Crr1基因序列新开发的多态性标记BrID10381不在新定位抗根肿基因初定位区间内,因此,可以推断新定位抗根肿基因位点与Crr1并非同一位点,且标记BrID10381可以用于Crr1基因的分子标记辅助选择。
In order to identify the clubroot resistance gene of Chinese cabbage in G6 and develop linkage markers,high-generation inbred line G4 of Chinese cabbage with high-susceptible to clubroot disease,high-generation inbred line G6 of Chinese cabbage with high-resistant to clubroot disease,F_1 obtained by crossing G4 and G6,and the F_2 generation separation populations constructed by F_1 selfing were used as materials. By artificial inoculation,phenotypic identification and genetic analysis,it was found that clubroot disease resistance in the disease resistance materials were controlled by a dominant single gene. Further expanding the number of F_(2 ) generation populations to map the genes of clubroot disease and screening molecular markers linked to disease resistance genes,the linkage analysis of polymorphic markers was performed using JoinMap 4.0 software,and five InDel markers linked to the resistance gene of Chinese cabbage were obtained. The most recent markers on both sides were BrID10727 and BrID10867,the genetic distance from the disease resistance genes were 4.6,2.5 cM,respectively,and the disease resistance gene was located on the chromosome A08 of Chinese cabbage. In addition,it was found that the newly developed polymorphic marker BrID10381 based on the Crr1 gene sequence was located outside the initial localization range of the newly located clubroot resistance gene,so it could be inferred that the newly located clubroot resistance gene locus was not the same locus as Crr1,and the marker BrID10381 could be used for molecular marker assisted selection of Crr1 gene.
In order to identify the clubroot resistance gene of Chinese cabbage in G6 and develop linkage markers,high-generation inbred line G4 of Chinese cabbage with high-susceptible to clubroot disease,high-generation inbred line G6 of Chinese cabbage with high-resistant to clubroot disease,F_1 obtained by crossing G4 and G6,and the F_2 generation separation populations constructed by F_1 selfing were used as materials. By artificial inoculation,phenotypic identification and genetic analysis,it was found that clubroot disease resistance in the disease resistance materials were controlled by a dominant single gene. Further expanding the number of F_(2 ) generation populations to map the genes of clubroot disease and screening molecular markers linked to disease resistance genes,the linkage analysis of polymorphic markers was performed using JoinMap 4.0 software,and five InDel markers linked to the resistance gene of Chinese cabbage were obtained. The most recent markers on both sides were BrID10727 and BrID10867,the genetic distance from the disease resistance genes were 4.6,2.5 cM,respectively,and the disease resistance gene was located on the chromosome A08 of Chinese cabbage. In addition,it was found that the newly developed polymorphic marker BrID10381 based on the Crr1 gene sequence was located outside the initial localization range of the newly located clubroot resistance gene,so it could be inferred that the newly located clubroot resistance gene locus was not the same locus as Crr1,and the marker BrID10381 could be used for molecular marker assisted selection of Crr1 gene.
引文
[1] 张凡,李林梅,崔海芳,尹俊龙,郭英琪,岳艳玲.十字花科植物抗根肿病研究进展[J].湖南生态科学学报,2017,4(3):57-62.doi:10.3969/j.issn.2095-7300.2017.03-057.Zhang F,Li L H,Cui H F,Yin J L,Guo Y Q,Yue Y Y.Current research of crucifer clubroot resistance[J].Journal of Hunan Environment Biological Polytechnic,2017,4(3):57-62.
[2] Kageyama K,Asano T.Life cycle of Plasmodiophora brassicae[J].Journal of Plant Growth Regulation,2009,28(3):203.doi:10.1007/s00344-009-9101-z.
[3] Zhang T,Zhao Z,Zhang C,Pang W X,Choi S R,Lin Y P,Piao Z Y.Fine genetic and physical mapping of the CRb gene conferring resistance to clubroot disease in Brassica rapa[J].Molecular Breeding,2014,34(3):1173-1183.doi:10.1007/s11032-014-0108-1.
[4] 董森森.白菜类作物抗根病种质资源遗传多样性分析及分子标记辅助选择[D].郑州:郑州大学,2014.Dong S S.Analysis of genetic diversity of the clubroot resistance germplasms in Brassica rapa and molecular marker-assisted selection[D].Zhengzhou:Zhengzhou University,2014.
[5] Nguyen M L,Monakhos G F,Komakhin R A,Monakhos S G.The new clubroot resistance locus is located on Chromosome A05 in Chinese cabbage(Brassica rapa L.)[J].Russian Journal of Genetics,2018,54(3):296-304.doi:10.1134/S1022795418030080.
[6] 胡靖锋,杨红丽,徐学忠,和江明,陈龙正.微型大白菜“娃娃菜”根肿病抗性遗传规律及分子标记辅助选择研究[J].福建农业学报,2017,32(2):185-188.doi:10.19303/j.issn.1008-0384.2017.02.016.Hu J F,Yang H L,Xu X Z,He J M,Chen L Z.Application of molecular markers-assisted selection for studying inheritance of clubroot-resistance in miniature chinese Baby cabbage [J].Fujian Journal of Agricultural Sciences,2017,32(2):185-188.
[7] 薛银鸽.大白菜根肿病抗性基因分子标记与定位[D].郑州:郑州大学,2014.Xue Y G.Molecular marker analysis and mapping of clubroot resistance gene in Chinese cabbage[D].Zhengzhou:Zhengzhou University,2014.
[8] 王彤彤,张淑江,章时蕃,李菲,张慧,孙日飞.大白菜根肿病抗性基因的标记和定位[J].中国蔬菜,2012,1(14):31-35.Wang T T,Zhang S J,Zhang S F,Li F,Zhang H,Sun R F.Mapping of clubroot resistance gene in Chinese cabbage[J].China Vegetables,2012,1(14):31-35.
[9] 王艳.白菜参考遗传图谱的构建[D].北京:中国农业科学院,2011.Wang Y.A sequence-based genetic linkage map as a reference for Brassica rapa L.ssp.pekinensis[D].Beijing:Chinese Academy of Agricultural Sciences,2011.
[10] 柴阿丽,朱发娣,王惟萍,石延霞,谢学文,李宝聚.十字花科根肿病接种技术及发病条件研究[J].华北农学报,2015,30(S1):266-271.doi:10.7668/hbnxb.2015.S1.048.Chai A L,Zhu F D,Wang W P,Shi Y X,Xie X W,Li B J.Inoculation methods and incidence conditions of clubroot of Cruciferous[J].Acta Agriculturae Boreali-Sinica,2015,30(S1):266-271.
[11] Pang W X,Liang S,Li X N,Li P P,Yu S,Lin Y P,Piao Z Y.Genetic detection of clubroot resistance loci in a new population of Brassica rapa[J].Horticulture,Environment,and Biotechnology,2014,55(6):540-547.doi:10.1007/s13580-014-0079-5.
[12] 董正中,陈超权,刘子萌,郑柳青,吴薇,谢涵冰,葛柳群,刘盛兰嘉,余小林.普通白菜形态学性状及其根肿病抗性鉴定[J].浙江农业科学,2017(11):140-143.doi:10.16178/j.issn.0528-9017.20171142.Dong Z Z,Chen C Q,Liu Z M,Zheng L Q,Wu W,Xie H B,Ge L Q,Liu S L J,Yu X L.The Morphological characters and identification of clubroot resistance of Brassica campestris(syn.B.rapa)L.ssp.chinensis(L)Makino[J].Zhejiang Agricultural Sciences,2017(11):140-143.
[13] 张红,张斌,王超楠,李梅,黄志银,刘俊峰.青麻叶大白菜根肿病人工接种方法及条件研究[J].华北农学报,2016,31(1):182-185.doi:10.7668/hbnxb.2016.01.029.Zhang H,Zhang B,Wang C N,Li M,Huang Z Y,Liu J F.Study on inoculation methods and condition of clubroot in Qingmaye Chinese cabbage[J].Acta Agriculturae Boreali-Sinica,2016,31(1):182-185.
[14] 王彤彤.大白菜根肿病抗性基因的分子标记及定位[D].北京:中国农业科学院,2012.Wang T T.Molecular marker analysis and mapping of clubroot resistance genes in Brassica rapa L.ssp.pekinensis[D].Beijing:Chinese Academy of Agricultural Sciences,2012.
[15] Piao Z Y,Deng Y Q,Choi S R,Park Y J,Lim Y P.SCAR and CAPS mapping of CRb,a gene conferring resistance to Plasmodiophora brassicae,in Chinese cabbage(Brassica rapa ssp.pekinensis)[J].Theoretical and Applied Genetics,2004,108(8):1458-65.
[16] Narzary D,Verma S,Mahar K S,Rana T S.A rapid and effective method for isolation of genomic DNA from small amount of silica-dried leaf tissues[J].National Academy Science Letters,2015,38(5):441-444.doi:10.1007/s40009-015-0357-5.
[17] Suwabe K,Tsukazaki H,Iketani H,Hatakeyama K,Kondo M,Fujimura M,Nunome T,Fukuoka H,Hirai M,Matsumoto S.Simple sequence repeat-based comparative genomics between Brassica rapa and Arabidopsis thaliana:the genetic origin of clubroot resistance[J].Genetics,2006,173(1):309-319.doi:10.1534/genetics.104.038968.
[18] Matsumoto E,Yasui C,Ohi M,Tsukada M.Linkage analysis of RFLP markers for clubroot resistance and pigmentation in Chinese cabbage[J].Euphytica,1998,104:79-86.doi:10.1023/a:1018370418201.
[19] Hayashida N,Takabatake Y,Nakazawa N,Aruga D,Nakanishi H,Taguchi G,Sakamoto K,Matsumoto E.Construction of a practical SCAR marker linked to clubroot resistance in Chinese cabbage with intensive analysis of HC352b genes[J].Japan Soc Hort Sci,2008,77(2):150-154.
[20] Hirai M,Harada T,Kubo N,Tsukada M,Suwabe K,Matsumoto S.A novel locus for clubroot resistance in,Brassica rapa,and its linkage markers[J].Theoretiscal and Applied Genetics,2004,108(4):639-643.doi:10.1007/s00122-003-1475-x.
[21] Sakamoto K,Saito A,Hayashida N,Taguchi G,Matsumoto E.Mapping of isolate-specific QTLs for clubroot resistance in Chinese cabbage(Brassica rapa L.ssp.pekinensis)[J].Theoretical and Applied Genetics,2008,117(5):759-767.doi:10.1007/s00122-008-0817-0.
[22] 任平平,马安峰,程斐,孙朝辉,高建伟.大白菜抗根肿病基因CRb的分子标记验证与种质资源筛选[J].山东农业科学,2016,48(6):7-10.doi:10.14083/j.issn,1001-4942.2016.06.002.Ren P P,Ma A F,Chen F,Sun C H,Gao J W.Identification of molecular markers for clubroot disease resistant gene cRb and screening of germplasm resources of Chinese cabbage[J].Shandong Agricultural Sciences,2016,48(6):7-10.
[23] Hatakeyama K,Niwa T,Kato T,Ohara T,Kakizaki T,Matsumoto S.The tandem repeated organization of NB-LRR genes in the clubroot-resistant CRb locus in Brassica rapa L.[J].Molecular Genetics and Genomics,2017,292(2):397-405.doi:10.1007/s00438-016-1281-1.
[24] Suwabe K,Tsukazaki H,Iketani H,Hatakeyama K,Fujimura M,Nunome T,Ukuoka H,Matsumoto S,Hirai M.Identification of two loci for resistance to clubroot(Plasmodiophora brassicae Woron.)in Brassica rapa L.[J].Theoretical and Applied Genetics,2003,107(6):997-1002.doi:10.1007/s00122-003-1309-x.
[25] Hatakeyama K,Suwabe K,Tomita R N,Kato T,Nunome T,Fukuoka H,Matsumoto S.Identification and characterization of crr1a,a gene for resistance to clubroot disease(Plasmodiophora brassicae Woron.)in Brassica rapa L.[J].PLoS One,2013,8(1):268-277.doi:10.1371/journal.pone.0054745.
[2] Kageyama K,Asano T.Life cycle of Plasmodiophora brassicae[J].Journal of Plant Growth Regulation,2009,28(3):203.doi:10.1007/s00344-009-9101-z.
[3] Zhang T,Zhao Z,Zhang C,Pang W X,Choi S R,Lin Y P,Piao Z Y.Fine genetic and physical mapping of the CRb gene conferring resistance to clubroot disease in Brassica rapa[J].Molecular Breeding,2014,34(3):1173-1183.doi:10.1007/s11032-014-0108-1.
[4] 董森森.白菜类作物抗根病种质资源遗传多样性分析及分子标记辅助选择[D].郑州:郑州大学,2014.Dong S S.Analysis of genetic diversity of the clubroot resistance germplasms in Brassica rapa and molecular marker-assisted selection[D].Zhengzhou:Zhengzhou University,2014.
[5] Nguyen M L,Monakhos G F,Komakhin R A,Monakhos S G.The new clubroot resistance locus is located on Chromosome A05 in Chinese cabbage(Brassica rapa L.)[J].Russian Journal of Genetics,2018,54(3):296-304.doi:10.1134/S1022795418030080.
[6] 胡靖锋,杨红丽,徐学忠,和江明,陈龙正.微型大白菜“娃娃菜”根肿病抗性遗传规律及分子标记辅助选择研究[J].福建农业学报,2017,32(2):185-188.doi:10.19303/j.issn.1008-0384.2017.02.016.Hu J F,Yang H L,Xu X Z,He J M,Chen L Z.Application of molecular markers-assisted selection for studying inheritance of clubroot-resistance in miniature chinese Baby cabbage [J].Fujian Journal of Agricultural Sciences,2017,32(2):185-188.
[7] 薛银鸽.大白菜根肿病抗性基因分子标记与定位[D].郑州:郑州大学,2014.Xue Y G.Molecular marker analysis and mapping of clubroot resistance gene in Chinese cabbage[D].Zhengzhou:Zhengzhou University,2014.
[8] 王彤彤,张淑江,章时蕃,李菲,张慧,孙日飞.大白菜根肿病抗性基因的标记和定位[J].中国蔬菜,2012,1(14):31-35.Wang T T,Zhang S J,Zhang S F,Li F,Zhang H,Sun R F.Mapping of clubroot resistance gene in Chinese cabbage[J].China Vegetables,2012,1(14):31-35.
[9] 王艳.白菜参考遗传图谱的构建[D].北京:中国农业科学院,2011.Wang Y.A sequence-based genetic linkage map as a reference for Brassica rapa L.ssp.pekinensis[D].Beijing:Chinese Academy of Agricultural Sciences,2011.
[10] 柴阿丽,朱发娣,王惟萍,石延霞,谢学文,李宝聚.十字花科根肿病接种技术及发病条件研究[J].华北农学报,2015,30(S1):266-271.doi:10.7668/hbnxb.2015.S1.048.Chai A L,Zhu F D,Wang W P,Shi Y X,Xie X W,Li B J.Inoculation methods and incidence conditions of clubroot of Cruciferous[J].Acta Agriculturae Boreali-Sinica,2015,30(S1):266-271.
[11] Pang W X,Liang S,Li X N,Li P P,Yu S,Lin Y P,Piao Z Y.Genetic detection of clubroot resistance loci in a new population of Brassica rapa[J].Horticulture,Environment,and Biotechnology,2014,55(6):540-547.doi:10.1007/s13580-014-0079-5.
[12] 董正中,陈超权,刘子萌,郑柳青,吴薇,谢涵冰,葛柳群,刘盛兰嘉,余小林.普通白菜形态学性状及其根肿病抗性鉴定[J].浙江农业科学,2017(11):140-143.doi:10.16178/j.issn.0528-9017.20171142.Dong Z Z,Chen C Q,Liu Z M,Zheng L Q,Wu W,Xie H B,Ge L Q,Liu S L J,Yu X L.The Morphological characters and identification of clubroot resistance of Brassica campestris(syn.B.rapa)L.ssp.chinensis(L)Makino[J].Zhejiang Agricultural Sciences,2017(11):140-143.
[13] 张红,张斌,王超楠,李梅,黄志银,刘俊峰.青麻叶大白菜根肿病人工接种方法及条件研究[J].华北农学报,2016,31(1):182-185.doi:10.7668/hbnxb.2016.01.029.Zhang H,Zhang B,Wang C N,Li M,Huang Z Y,Liu J F.Study on inoculation methods and condition of clubroot in Qingmaye Chinese cabbage[J].Acta Agriculturae Boreali-Sinica,2016,31(1):182-185.
[14] 王彤彤.大白菜根肿病抗性基因的分子标记及定位[D].北京:中国农业科学院,2012.Wang T T.Molecular marker analysis and mapping of clubroot resistance genes in Brassica rapa L.ssp.pekinensis[D].Beijing:Chinese Academy of Agricultural Sciences,2012.
[15] Piao Z Y,Deng Y Q,Choi S R,Park Y J,Lim Y P.SCAR and CAPS mapping of CRb,a gene conferring resistance to Plasmodiophora brassicae,in Chinese cabbage(Brassica rapa ssp.pekinensis)[J].Theoretical and Applied Genetics,2004,108(8):1458-65.
[16] Narzary D,Verma S,Mahar K S,Rana T S.A rapid and effective method for isolation of genomic DNA from small amount of silica-dried leaf tissues[J].National Academy Science Letters,2015,38(5):441-444.doi:10.1007/s40009-015-0357-5.
[17] Suwabe K,Tsukazaki H,Iketani H,Hatakeyama K,Kondo M,Fujimura M,Nunome T,Fukuoka H,Hirai M,Matsumoto S.Simple sequence repeat-based comparative genomics between Brassica rapa and Arabidopsis thaliana:the genetic origin of clubroot resistance[J].Genetics,2006,173(1):309-319.doi:10.1534/genetics.104.038968.
[18] Matsumoto E,Yasui C,Ohi M,Tsukada M.Linkage analysis of RFLP markers for clubroot resistance and pigmentation in Chinese cabbage[J].Euphytica,1998,104:79-86.doi:10.1023/a:1018370418201.
[19] Hayashida N,Takabatake Y,Nakazawa N,Aruga D,Nakanishi H,Taguchi G,Sakamoto K,Matsumoto E.Construction of a practical SCAR marker linked to clubroot resistance in Chinese cabbage with intensive analysis of HC352b genes[J].Japan Soc Hort Sci,2008,77(2):150-154.
[20] Hirai M,Harada T,Kubo N,Tsukada M,Suwabe K,Matsumoto S.A novel locus for clubroot resistance in,Brassica rapa,and its linkage markers[J].Theoretiscal and Applied Genetics,2004,108(4):639-643.doi:10.1007/s00122-003-1475-x.
[21] Sakamoto K,Saito A,Hayashida N,Taguchi G,Matsumoto E.Mapping of isolate-specific QTLs for clubroot resistance in Chinese cabbage(Brassica rapa L.ssp.pekinensis)[J].Theoretical and Applied Genetics,2008,117(5):759-767.doi:10.1007/s00122-008-0817-0.
[22] 任平平,马安峰,程斐,孙朝辉,高建伟.大白菜抗根肿病基因CRb的分子标记验证与种质资源筛选[J].山东农业科学,2016,48(6):7-10.doi:10.14083/j.issn,1001-4942.2016.06.002.Ren P P,Ma A F,Chen F,Sun C H,Gao J W.Identification of molecular markers for clubroot disease resistant gene cRb and screening of germplasm resources of Chinese cabbage[J].Shandong Agricultural Sciences,2016,48(6):7-10.
[23] Hatakeyama K,Niwa T,Kato T,Ohara T,Kakizaki T,Matsumoto S.The tandem repeated organization of NB-LRR genes in the clubroot-resistant CRb locus in Brassica rapa L.[J].Molecular Genetics and Genomics,2017,292(2):397-405.doi:10.1007/s00438-016-1281-1.
[24] Suwabe K,Tsukazaki H,Iketani H,Hatakeyama K,Fujimura M,Nunome T,Ukuoka H,Matsumoto S,Hirai M.Identification of two loci for resistance to clubroot(Plasmodiophora brassicae Woron.)in Brassica rapa L.[J].Theoretical and Applied Genetics,2003,107(6):997-1002.doi:10.1007/s00122-003-1309-x.
[25] Hatakeyama K,Suwabe K,Tomita R N,Kato T,Nunome T,Fukuoka H,Matsumoto S.Identification and characterization of crr1a,a gene for resistance to clubroot disease(Plasmodiophora brassicae Woron.)in Brassica rapa L.[J].PLoS One,2013,8(1):268-277.doi:10.1371/journal.pone.0054745.